Maintenance of cholesterol and fatty acid homeostasis is critical for membrane architecture, protein localization and trafficking, and cellular function. Dysregulation can lead to severe health concerns including obesity, diabetes, heart attack and stroke. Vital to the management of lipid molecules are the sterol regulatory element binding protein (SREBP) family of transcription factors. This family of three proteins transcribes more than 30 genes that control lipid homeostasis. The activation of SREBP is tightly regulated through association with the SREBP cleavage-activating protein (SCAP). SCAP and SREBP bind through their respective C-terminal domains (CTD), but the affinity, stoichiometry, and interface are not known. When sterol levels are high, SCAP maintains SREBP in the endoplasmic reticulum in an inactive form. As sterol concentrations are reduced, SCAP escorts SREBP to the Golgi apparatus where it is activated. Liver-specific knockout of SCAP results in a 70-80% decrease in lipid biosynthesis and germline knockout is hypothesized to be embryonic lethal. Nonetheless, how SCAP recognizes and restricts the location of SREBP is relatively unknown. As a single SCAP variant manages all three SREBP proteins it is reasonable to suspect it may provide an additional level of signaling regulation. Therefore, the overall goal of this proposal is to determine the atomic details of the SCAP CTD, SREBP CTD, and characterize the binding interface. The SCAP CTD contains seven WD40 repeat motifs that are speculated to form a seven-bladed beta-propeller. However, sequence comparison to known structures suggests the existence of an eighth blade following the last WD40 repeat. Aim 1 will test the hypothesis that the SCAP CTD forms an eight-bladed beta-propeller structure. In addition to determining the protein fold and presenting putative binding interfaces, the SCAP CTD would constitute the first NMR characterization of a beta-propeller structure and may reveal general conformational dynamics of this fold. Aim 2 will use a combined approach of NMR and biochemical assays to test the hypothesis that the SREBP-1c CTD possesses a stable tertiary structure in the absence of SCAP. Cell-based assays indicate that the first 200 amino acids of the CTD are sufficient for SCAP binding, but with a reduced affinity compared to the full- length CTD. Our preliminary sequence analysis suggests these residues form a contiguous globular fold followed by two additional ordered regions. The resulting structural and dynamic information will suggest how the full-length and truncated forms can recognize SCAP and may also suggest how post-translational modifications affect complex formation. In aim 3 we will pursue the first characterization of a SCAP/SREBP-1c complex to identify the affinity, stoichiometry, and residues important for binding. We propose to use a combined NMR, biochemical, and cell-based approach to elucidate the thermodynamic, kinetic, and structural parameters underlying this interaction. Our results may also provide a structural basis for abnormal SREBP activity and benefit drug discovery efforts aimed at inhibiting aberrant SREBP signaling. PUBLIC HEALTH RELEVANCE: Interactions between the proteins SCAP and SREBP are crucial to maintaining healthy cholesterol and fatty acid concentrations. Dysregulation of these proteins can lead to obesity, diabetes, heart disease and stroke. This proposal aims to determine the molecular details of this interaction, a step that is crucial to the development of therapeutics drugs.